Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 189

Fragmentation agent
(e.g. trypsin)

(a)

5

Chromatography

4

2

3

1

Origin

Electrophoresis

(b)

Figure 7.4 Generation of a peptide map. In this simple example, the protein to be analysed is treated with
a fragmentation agent, e.g. trypsin (a). In this case, fi ve fragments are generated. The digest is then ap-
plied to a sheet of chromatography paper (b) (at the point marked 'origin'). The peptides are then separated
from each other in the fi rst (vertical) dimension by paper chromatography. Subsequently, electrophoresis is
undertaken (in the horizontal direction). The separated peptide fragments may be visualized by, for example,
staining with ninhydrin. Two-dimensional separation of the peptides is far more likely to resolve each peptide
completely from the others. In the case above, for example, chromatography (in the vertical dimension) alone
would not have been suffi cient to resolve peptides 1 and 3 fully. During biopharmaceutical production, each
batch of the recombinant protein produced should yield identical peptide maps. Any mutation that alters
the protein's primary structure (i.e. amino acid sequence) should result in at least one fragment adopting an
altered position in the peptide map

this way, single (or multiple) amino acid substitutions, deletions, insertions or modifications
can usually be detected. This technique plays an important role in monitoring batch-to-
batch consistency of the product, and also obviously can confirm the identity of the actual
product.
The choice of reagent used to fragment the protein is critical to the success of this approach.
If a reagent generates only a few very large peptides, a single amino acid alteration in one such
peptide will be more diffi cult to detect than if it occurred in a much smaller peptide fragment.
On the other hand, generation of a large number of very short peptides can be counterproduc-
tive, as it may prove diffi cult to resolve all the peptides from each other by subsequent chroma-
tography. Generation of peptide fragments containing an average of 7-14 amino acids is most
desirable.
The most commonly utilized chemical cleavage agent is cyanogen bromide (it cleaves the pep-
tide bond on the carboxyl side of methionine residues). V8 protease, produced by certain staphy-
lococci, along with trypsin are two of the more commonly used proteolytic-based fragmentation
agents.